Systems and Methods for Biomimetic Fluid Processing

ABSTRACT

Systems and methods generating physiologic models that can produce functional biological substances are provided. In some aspects, a system includes a substrate and a first and second channel formed therein. The channels extend longitudinally and are substantially parallel to each other. A series of apertures extend between the first channel and second channel to create a fluid communication path passing through columns separating the channels that extends further along the longitudinal dimension than other dimensions. The system also includes a first source configured to selectively introduce into the first channel a first biological composition at a first channel flow rate and a second source configured to selectively introduce into the second channel a second biological composition at a second channel flow rate, wherein the first channel flow rate and the second channel flow rate create a differential configured to generate physiological shear rates within a predetermined range in the channels.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on, claims the benefit of, and incorporates byreference U.S. Provisional Application No. 61/972,520 filed Mar. 31,2014, and entitled “SYSTEM AND METHOD FOR BIOMIMETIC FLUID PROCESSING.”

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This disclosure was made with government support under 1K99HL114719-01A1and HL68130 awarded by the National Institutes of Health. The governmenthas certain rights in the disclosure.

BACKGROUND OF THE DISCLOSURE

The present disclosure generally relates to fluid systems, includingmicrofluidic devices, systems that include such devices, and methodsthat use such devices and systems. More particularly, the presentdisclosure relates to devices, systems, and methods for producingfunctional biological material, substances, or agents based onbiomimetic platforms.

Blood platelets (PLTs) are essential for hemostasis, angiogenesis, andinnate immunity, and when numbers dip to low levels, a condition knownas thrombocytopenia, a patient is at serious risk of death fromhemorrhage. Some causes for low platelet count include surgery, cancer,cancer treatments, aplastic anemia, toxic chemicals, alcohol, viruses,infection, pregnancy, and idiopathic immune thrombocytopenia.

Replacement PLTs to treat such conditions are generally derived entirelyfrom human donors, despite serious clinical concerns owing to theirimmunogenicity and associated risk of sepsis. However, the shortagescreated by increased demand for PLT transfusions, coupled withnear-static pool of donors and short shelf-life on account of bacterialtesting and deterioration, are making it harder for health careprofessionals to provide adequate care for their patients. Moreover,alternatives such as artificial platelet substitutes, have thus farfailed to replace physiological platelet products.

In vivo, PLTs are produced by progenitor cells, known as megakaryocytes(MKs), in a process illustrated in FIG. 8. Located outside blood vesselsin the bone marrow (BM), MKs extend long, branching cellular structures(proPLTs) into sinusoidal blood vessels, where they experience shearrates and release PLTs into the circulation. While functional human PLTshave been grown in vitro, cell culture approaches to-date have yieldedonly about 10 percent proPLT production and 10-100 PLTs per human MK. Bycontrast, nearly all adult MKs in humans must produce roughly1,000-10,000 PLTs each to account for the number of circulating PLTs.This constitutes a significant bottleneck in the ex vivo production ofplatelet transfusion units.

In addition, although second generation cell culture approaches haveprovided further insight into the physiological drivers of PLT release,they have been unable to recreate the entire BM microenvironment,exhibiting limited individual control of extracellular matrix (ECM)composition, BM stiffness, endothelial cell contacts, or vascular shearrates; and have been unsuccessful in synchronizing proPLT production,resulting in non-uniform PLT release over a period of 6-8 days.Moreover, the inability to resolve pro PLT extension and release underphysiologically relevant conditions by high-resolution live-cellmicroscopy has significantly hampered efforts to identify thecytoskeletal mechanics of PLT production to enable drug development andestablish new treatments for thrombocytopenia. Therefore, an efficient,donor-independent PLT system capable of generating clinicallysignificant numbers of functional human PLTs is necessary to avoid risksassociated with PLT procurement and storage, and help meet growingtransfusion needs.

Considering the above, there continues to be a clear need for devices,systems, and methods employing platforms that can reproduce vascularphysiology in order to accurately reflect the processes, mechanisms, andconditions influencing the efficient production of functional humanblood platelets. Such platforms would prove highly useful for thepurposes of efficiently generating human platelets for infusion, as wellas for establishing drug effects and interactions in the preclinicalstages of development.

SUMMARY OF THE DISCLOSURE

The present disclosure overcomes the drawbacks of aforementionedtechnologies by providing systems and methods capable of producingphysiologically accurate models, which replicate conditions,environments, structures, and dynamic flows found in vivo. As will bedescribed, such physiological models may be utilized to generatefunctional human blood platelets, and other biological materials, thatwould be amenable for infusive treatment of certain medical conditions,such as platelet-deficient conditions like thrombocytopenia. Otherapplications for physiological models, produced in accordance with thepresent disclosure, may also include drug development, as well as drugand treatment assessment.

In accordance with one aspect of the present disclosure, a biomimeticmicrofluidic system is provided. The system includes a substrate, afirst channel formed in the substrate, the first channel extending froma first input to a first output along a longitudinal dimension andextending along a first transverse dimension, and a second channelformed in the substrate, the second channel extending from a secondinput to a second output along the longitudinal dimension and extendingalong the first transverse dimension, wherein the first and secondchannels extend substantially parallel along the longitudinal dimensionand are separated by columns extending along a second transversedimension. The system also includes a series of apertures formed in thecolumns separating the first channel and second channel, wherein each ofthe series of apertures extend along the longitudinal dimension furtherthan in the first transverse direction and the second transversedirection and are positioned proximal to a first portion of thesubstrate and extend from the first channel to the second channel tocreate a fluid communication path passing between the first channel andsecond channel. The system further includes a first source connected tothe first input, the first source configured to selectively introduceinto the first channel at least one first biological composition at afirst channel flow rate, and a second source connected to the secondinput, the second source configured to selectively introduce into thesecond channel at least one second biological composition at a secondchannel flow rate, wherein the first channel flow rate and the secondchannel flow rate create a differential configured to generatephysiological shear rates within a predetermined range in the channels.

In another aspect of the present disclosure, a method is disclosed forproducing a physiological model of at least one of a bone marrow andblood vessel structure. A method includes providing a biomimeticmicrofluidic system that includes a substrate, a first channel formed inthe substrate, the first channel extending from a first input to a firstoutput along a longitudinal dimension and a first transverse dimension.A second channel is formed in the substrate, the second channelextending from a second input to a second output along the longitudinaldimension and the first transverse dimension. A third channel is formedin the substrate, the third channel extending from the second input to athird output along the longitudinal dimension and the first transversedimension, wherein the first, second, and third channels extendsubstantially parallel along the longitudinal dimension and extend alonga second transverse dimension. A series of microchannels connect thefirst channel to the second channel and connecting the third channel tothe first channel, wherein the series of microchannels extend further inthe longitudinal dimension than the first transverse direction and thesecond transverse direction and is positioned proximal to a firstportion of the substrate to create a fluid communication path passingbetween the first channel and the second channel and the first channeland the third channel proximate to the first portion of the substrate.The method also includes introducing a first biological composition intothe first channel at a first channel flow rate using the first source,and introducing a second biological composition into the second channeland third channel using the second source and at a second channel flowrate and a third channel flow rate, respectively, to create adifferential between the first, second and third channel flow rates togenerate physiological shear rates within a predetermined range in thechannels. The method further includes harvesting a target biologicalsubstance produced proximate to the microchannels by the physiologicalshear rates.

In yet another aspect of the present disclosure, another biomimeticmicrofluidic system is provided. The system includes at least onesubstrate, and a first chamber formed in the at least one substrate, thefirst chamber extending from a first input to a first outputsubstantially along a longitudinal dimension. The system also includes asecond chamber formed in the at least one substrate, the second chamberextending from a second input to a second output along the longitudinaldimension, wherein the first and second chambers extend substantiallyparallel along the longitudinal dimension, and a membrane separating thefirst and second chamber along a transverse dimension, wherein themembrane creates a fluid communication path passing between the firstchamber and second chamber. The system further includes at least onesource configured to selectively introduce into the first chamber andthe second chamber, using respective inputs, at least one biologicalcomposition at flow rates capable of generating physiological shearrates between the chambers that facilitate production of a pluralityblood platelets.

The foregoing and other aspects and advantages of the disclosure willappear from the following description. In the description, reference ismade to the accompanying drawings which form a part hereof, and in whichthere is shown by way of illustration a preferred embodiment of thedisclosure. Such embodiment does not necessarily represent the fullscope of the disclosure, however, and reference is made therefore to theclaims and herein for interpreting the scope of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure will hereafter be described with reference to theaccompanying drawings, wherein like reference numerals denote likeelements.

FIG. 1 shows a schematic diagram of an example biomimetic microfluidicsystem, in accordance with aspects of the present disclosure.

FIG. 2A shows microscopy images depicting microfluidic channels coatedwith bone marrow and blood vessel proteins for reproducingextra-cellular matrix (ECM) composition.

FIG. 2B shows microscopy images depicting megakaryocytes (MKs) trappedin microchannels selectively embedded in alginate gel, modeling3-dimensional ECM organization and physiological bone marrow (BM)stiffness.

FIG. 2C shows microscopy images of human umbilical vein endothelialcells (HUVECs) selectively cultured in the fibrinogen-coated secondchannel to produce a functional blood vessel.

FIG. 2D shows an image of a complete system for producing functionalblood platelets (PLTs).

FIG. 2E shows a graphical depiction of a simulated distribution of shearrates within a biomimetic microfluidic system in accordance with aspectsthe present disclosure.

FIG. 2F shows a graph showing shear rates as a function of transversedistance from first channel for several infusion rates.

FIG. 2G shows a graph showing shear rates as a function of the number ofblock microchannels.

FIG. 3A shows a graph showing diameter distribution of cultured MKs at 0and 18 hours.

FIG. 3B shows microscopy images of MKs in static culture illustratingproduction of proPLTs at 6 hours post-purification.

FIG. 3C shows a microscopy image showing the production proPLT underphysiological shear immediately upon MK trapping.

FIG. 3D shows a graph showing increased proPLT-producing MKs obtainedunder physiological shear versus those from static cultures.

FIG. 3E shows a graph showing proPLT extension rates under physiologicalshear.

FIG. 4A shows microscopy images that illustrate MKs squeezing through 3μm-wide microchannels.

FIG. 4B shows microscopy images that illustrate MKs extending largefragments through 3 μm-wide microchannels.

FIG. 4C shows microscopy images that illustrate proPLT extension.

FIG. 4D shows microscopy images that illustrate proPLT extension andabscission events at different positions along the proPLT shaft.

FIG. 4E shows microscopy images that illustrate the cycle of PLTproduction.

FIG. 4F shows a graph showing that increased shear rates withinphysiological ranges do not increase proPLT extension rate.

FIG. 4G shows a microscopy image showing that MKs, retrovirallytransfected to express GFP-β1 tubulin, showed proPLT extensions andincluded peripheral microtubules that form coils at the PLT-sized ends.

FIG. 4H shows a graph illustrating that 5 βM Jasplankinolide (Jas, actinstabilizer) and 1 mM erythro-9-(3-[2-hydroxynonyl] (EHNA, cytoplasmicdynein inhibitor) inhibit shear-induced proPLT production.

FIG. 4I shows microscopy images that illustrate drug-induced inhibitionof proPLT production under physiological shear.

FIG. 5A shows graphs illustrating that PLTs produced in accordance withthe present disclosure manifest structural and functional properties ofblood PLTs.

FIG. 5B shows graphs that illustrate biomarker expression, forward/sidescatter and relative concentration of GPIX+MKs infused into a system, inaccordance with the present disclosure, following isolation from cultureon day 4, and collection from effluent 2 hours post infusion.

FIG. 5C shows a graph illustrating that the application of shear shiftsGPIX+ produce more PLT-sized cells relative to static culturesupernatant.

FIG. 5D shows microscopy images illustrating that MKs are converted intoPLTs over a period of 2 hours.

FIG. 5E is a graphical illustration showing that application of shearshifts produce more PLT-sized β1 tubulin+ Hoescht− cells relative tostatic culture supernatant, with the insert shows quantitation of freenuclei in the effluent.

FIG. 5F shows microscopy images illustrating that PLTs, produced inaccordance with the present disclosure, are ultrastructurally similar toblood PLTs and contain a cortical MT coil, open canalicular system,dense tubular system, mitochondria, and characteristic secretorygranules.

FIG. 5G shows microscopy images that illustrate PLTs and PLTintermediates, produced in accordance with the present disclosure, aremorphologically similar to blood PLTs and display comparable MT andactin expression.

FIG. 6A shows a graph illustrating that hiPSC-PLTs, derived inaccordance with the present disclosure, manifest structural andfunctional properties of blood PLTs.

FIG. 6B shows a microscopy image illustrating that hiPSC-MKs, inaccordance with the present disclosure, are ultrastructurally similar toprimary human MKs and contain a lobulated nuclei, invaginated membranesystem, glycogen stores, organelles, and characteristic secretorygranules.

FIG. 6C shows microscopy images illustrating that hiPSC-MKs in staticculture begin producing proPLTs at 6 hours post-purification, and reachmaximal proPLT production at 18 hours.

FIG. 6D shows a microscopy image illustrating that hiPSC-MKs underphysiological shear (about 500 s⁻¹) begin producing proPLTs immediatelyupon trapping and extend/release proPLTs within the first 2 hours ofculture.

FIG. 6E is a graphic illustrating that percent proPLT-producinghiPSC-MKs under physiological shear are increased significantly overstatic cultures.

FIG. 6F is a graph illustrating that proPLT extension rates underphysiological shear are about 19 μm/min.

FIG. 6G shows microscopy images illustrating that hPLTs, derived inaccordance with the present disclosure ,are ultrastructurally similar tohuman blood PLTs and contain a cortical MT coil, open canalicularsystem, dense tubular system, mitochondria, and characteristic secretorygranules.

FIG. 6H shows microscopy images illustrating that hPLTs, derived inaccordance with the present disclosure, are morphologically similar tohuman blood PLTs and display comparable MT and actin expression.

FIG. 6I shows microscopy images illustrating that mPLTs, derived inaccordance with the present disclosure, form filpodia/lamellipodia onactivation and spread on glass surface.

FIG. 7 shows live-cell microscopy images illustrating that T-DM1inhibits MK differentiation and disrupts proPLT formation by inducingabnormal tubulin organization.

FIG. 8 shows an illustration depicting PLT production in vivo.

FIG. 9 shows a schematic diagram of another example biomimeticmicrofluidic system, in accordance with aspects of the presentdisclosure.

FIG. 10 shows an illustration depicting PLT production using the systemof FIG. 9.

FIG. 11A shows an illustration comparing PLT production using one fluidflow implementation in the system of FIG. 9.

FIG. 11B shows an illustration comparing PLT production using anotherfluid flow implementation in the system of FIG. 9.

FIG. 12 shows an illustration of yet another biomimetic microfluidicsystem, in accordance with aspects of the present disclosure.

FIG. 13 is a flowchart setting forth steps of a process for producing aphysiological model, in accordance with aspects of the presentdisclosure.

DETAILED DESCRIPTION OF THE DISCLOSURE

Blood platelets (PLTs) play a critical role in stimulating clotformation and repair of vascular injury. Morbidity and mortality frombleeding due to low PLT count is a major clinical problem encounteredacross a number of conditions including chemotherapy or radiationtreatment, trauma, immune thrombocytopenic purpura (ITP), organtransplant surgery, severe burns, sepsis, and genetic disorders. Despiteserious clinical concerns owing to their immunogenicity and associatedrisks, along with inventory shortages due to high demand and short shelflife, PLT transfusions total more than 10 million units per year in theUnited States.

In recognizing such wide-spread needs and risks, the present disclosuredescribes herein systems and methods capable of replicating conditions,environments, structures, and dynamic flows present in physiology. Suchsystems and methods may be used to generate models of human physiology,which can then be used to produce functional human PLTs, for instance.

PLT production involves the differentiation of megakaryocyte (MKs),which sit outside sinusoidal blood vessels in the bone marrow (BM) andextend long, branching cellular structures (designated proPLTs) into thecirculation, as shown in FIG. 8. Specifically, proPLTs include PLT-sizedswellings in tandem arrays connected by thin cytoplasmic bridges. Invivo, they experience vascular shear and function as the assembly linesfor PLT production. Although detailed characterization of proPLTsremains incomplete, these structures have been identified both in vitroand in vivo.

PLTs are released sequentially from proPLT tips. This mechanism ishighly dependent on a complex network of tubulin and actin filamentsthat function as the molecular struts and girders of a cell. Microtubule(MT) bundles run parallel to proPLT shafts, and proPLT longation isdriven by MTs sliding over one another. During proPLT maturation,organelles and secretory granules traffic distally over MT rails tobecome trapped at proPLT tips. Actin promotes proPLT branching andamplification of PLT ends. Live cell microscopy of murine MKs has beenvital to this understanding, however most studies to date have been donein vitro on static MK cultures.

Thrombopoietin (TPO) has been identified as the major regulator of MKdifferentiation, and it has been used to produce enriched populations ofMKs in culture. In one reference, it was demonstrated that human PLTsgenerated in vitro from proPLT-producing MKs were functional. Sincethen, MKs have been differentiated from multiple sources, includingfetal liver cells (FLCs), cord blood stem cells (CBSCs), embryonic stemcells (ESCs), and induced pluripotent stem cells (iPSCs). However,current 2-D and liquid MK cultures fall orders of magnitude short of theestimated about 2000 PLTs generated per MK in vivo. More recently,modular 3-D knitted polyester scaffolds have been applied undercontinuous fluid flow to produce up to 6×10⁶ PLTs/day from 1 millionCD34⁺ human cord blood cells in culture. While suggestive thatclinically useful PLT numbers may be attained, those 3-D perfusionbioreactors do not accurately reproduce the complex structure and fluidcharacteristics of the BM microenvironment, and their closed modulardesign prevents visualization of proPLT production, offering littleinsight into the mechanism of PLT release. Alternatively, 3-Dpolydimethylsiloxane (PDMS) biochips adjacent ECM-coated silk-basedtubes have been proposed to reproduce BM sinusoids and study MKdifferentiation and PLT production in vitro. Although such devicesrecapitulate MK migration during maturation, they are not amenable tohigh resolution live-cell microscopy, and fail to reproduce endothelialcell contacts necessary to drive MK differentiation.

While MK differentiation has been studied in culture, the conditionsthat stimulate proPLT production remain poorly understood, particularlyin vivo. MKs are found in BM niches, and some evidence suggests thatcell-cell, cell-matrix, and soluble factor interactions of the BM stromacontribute to proPLT formation and PLT release. Indeed, the chemokineSDF-1 and growth factor FGF-4 recruit MKs to sinusoid endothelial cells.Extracellular matrix (ECM) proteins are another major constituent of theBM vascular niche, and evidence suggests that signaling throughtrans-membrane glycoprotein (GP) receptors regulate proPLT formation,PLT number and size, with defects seen for example, in Bernard-Souliersyndrome, Glanzmann's thrombasthenia. Collagen IV and vitronectinpromote proPLT production, which can be inhibited by antibodies directedagainst their conjugate integrin receptor, GPIbα. Likewise, fibrinogenregulates proPLT formation and PLT release through GPIIbIIIa. Whilethese findings shed light on the environmental determinants of proPLTproduction, they are limited by a reductionist approach. Therefore, newmodels that incorporate the defining attributes of BM complexity arenecessary to elucidate the physiological regulation of MKs into PLTs.

In the BM, proPLTs experience wall shear rates ranging from, 100 to10,000 s⁻¹ or, more particularly, from 500 to 2500 s⁻¹. While the roleof continuous blood flow on PLT thrombus formation has been studied,surprisingly little attention has been paid to the mechanism by whichshear forces regulate PLT release. Also, when investigated, experimentshave not been representative of true physiological conditions. Somepreliminary studies have perfused MKs over ECM-coated glass slides,which select for immobilized/adhered MKs without discriminatingECM-contact activation from shear. Alternatively, released proPLTs havebeen centripetally agitated in an incubator shaker, which does notrecapitulate circulatory laminar shear flow, does not provide precisecontrol of vascular shear rates, and is not amenable to high-resolutionlive-cell microscopy. Despite these major limitations, exposure of MKsto high shear rates appears to accelerate proPLT production and whileproPLTs cultured in the absence of shear release fewer PLTs than thosemaintained at fluid shear stresses.

The present disclosure recognizes that microfluidic devices can provideexcellent platforms to generate and precisely tune dynamic fluid flows,and thus mimic blood vessel conditions for delivering chemical cues tocells. Embedding microfluidic networks within cell-laden hydrogels hasbeen shown to support efficient convective transport of soluble factorsthrough 3D scaffolds. Viable 3D tissue contacts have been producedconsisting of hepatocytes encapsulated in agarose, calcium alginatehydrogels seeded with primary chondrocytes, and endothelial cellsembedded in 3D tubular poly(ethylene glycol) hydrogels. Accordingly, thetechnology has been applied to the development of organs-on-a-chip,including liver, kidney, intestine, and lung. In addition, recentdevelopment of microvasculature-on-a-chip models have been used to studycardiovascular biology and pathophysiology in vitro. These studiesemphasize the importance of mimicking the physical microenvironment andnatural chemical cues of living organs to study cellular andphysiological development. For example, this is particularly importantfor drug-mediated inhibition of PLT production. Since proPLT-producingMKs sit just outside blood vessels in the BM, interacting with both thesemi-solid ECM microenvironment of BM and fluid microenvironment of thecirculation, biomimetic microfluidic biochips may achieve a model systemto elucidating the relevant physiological mechanisms, such as thoseresponsible for drug-induced thrombocytopenia.

Turning now to FIG. 1, a schematic is shown illustrating a non-limitingexample of a biomimetic system 100, in accordance with variousembodiments of the present disclosure. The system 100 includes asubstrate 101, a first channel 102 and a second channel 104, whereineach channel is configured to carry a flow of any fluid mediumtransporting or consisting of but not limited to, for example,particles, cells, substances, particulates, materials, compositions andthe like. In one embodiment, the system 100 and/or substrate 101 may beconstructed using cell-inert silicon-based organic polymers, such aspolydimethylsiloxane (PDMS).

The first channel 102 includes a first channel input 106 and firstchannel output 108. Similarly, the second channel 104 includes a secondchannel input 110 and second channel output 112. The first channel 102and second channel 104 extend along a substantially longitudinaldirection, and are longitudinally and transversally dimensioned, as willbe explained, to achieve desired flow profiles, velocities, or rates,such as those present in a physiological system. In one aspect, the sizeof the longitudinal 130 and transverse 132 dimensions describing thechannels may be in a range consistent with an anatomical orphysiological structure, assembly, configuration or arrangement, such asin the case of bone marrow and blood vessels. By way of example, thelongitudinal 130 dimension may be in the range of 1000 to 30,000micrometers or, more particularly, in the range of 1000 to 3000micrometers, and the transverse 132 dimension may be in the range of 100to 3,000 micrometers or, more particularly, in the range of 100 to 300micrometers, although other values are possible. In some aspects, eachchannel may be prepared, conditioned, or manufactured to receive,localize, trap, or accumulate for example, particles, cells, substances,particulates, materials, compositions, and the like, from a traversingfluid medium.

The system 100 also includes a series of columns 114 that separate thefirst channel 102 and second channel 104. The long axes of the columns114 may be arranged substantially parallel to the longitudinal 130dimension of the channels, the series of columns 114 extending for adistance substantially equal to the longitudinal 130 dimension of thechannels. The columns 114 may be separated by a series of gaps, ormicrochannels 116, that extend from the first channel 102 to the secondchannel 104 to create a partial fluid communication path passing betweenthe columns 114. However, the term “microchannel” when referring to thegaps does not connote a particular width. For example, the gaps may besubstantially greater or smaller than the micrometer range. In someaspects, the columns 114 and microchannels 116 may be dimensioned suchthat particles, cells, substances, particulates, materials,compositions, and the like, may bind, adhere to or otherwise be confinedto an area generally in the vicinity of the columns 114 andmicrochannels 116. As an example, the longitudinal 130 and transverse132 dimensions of the columns 114 may be in the range of 1 to 200micrometers, while the longitudinal 130 dimension of the microchannels116, defined by the separation distances or gaps between the columns114, may be in the range of 0.1 to 20 micrometers, although other valuesare possible.

In some aspects, flow of a first medium in the first channel 102 may beestablished using a first source coupled to a first inlet 118, whereinthe flow of the first medium is extractable via a first outlet 120.Similarly, flow of a second medium in the second channel 102 may beestablished using a second source coupled to a second inlet 118, whereinthe flow of the second medium is extractable via a second outlet 120.However, flow from the first channel 102 or second channel 104 may beextracted through either the first outlet 120 or the second outlet 122by virtue of the fluid communication between them. In someconfigurations, either the first outlet 120 or the second outlet 124, orboth, may include capabilities for draining or capturing flowestablished using the first source or the second source, or both. Suchcapabilities may also include the ability to separate a desired materialor substance from captured flow, such as blood platelets orthrombocytes.

The first and second source (not shown in FIG. 1) may include any systemconfigured for delivering a controlled flow or fluid pressure, such amicrofluidic pump system, and include any number of elements orcomponents, such as flow regulators, actuators, and so forth. Flowvelocities or flow rates, sustainable for any desired or required amountof time, may be controlled using specific configuration of sources,elements and components, as well as by virtue of the geometricaldimensions associated with the first channel 102 and second channel 104.In some aspects, flow rates in the first channel 102 and second channel104 may be controlled to duplicate physiological conditions, as found,for example, in bone marrow and blood vessels. For instance, flow ratesmay be controlled to achieve desired vascular shear rates sufficient forgenerating PLTs.

The system 100 may also include filtration and resistive elements, ofany shape or form, and arranged along the paths of each of the first andsecond fluid mediums in dependence of the direction of flow.Specifically, the filtration elements may be designed to capture orremove from the traversing fluid mediums any kind of debris, dust andother contaminants or undesirable materials, elements, or particulates.In addition, the resistive elements may be desired to control flowforces or damp fluctuations in flow rate. In some configurations, asshown in FIG. 1, filtration elements 126 may be situated in proximity tothe first inlet 118 and second inlet 122 in order to immediately captureundesired contaminants. The flow resistive elements 128 may then besituated downstream from the filtration elements 126, as shown in FIG.1.

In some implementations, recreating human bone marrow (BM) vascularniche ex vivo may be achieved by selectively filling the first channel102 with any combination of bone powder, peptides, or proteins thatregulate platelet producing, including but not limited to CI, CIV, FG,FN, VN, LN, VWF Poly-L-lysine, fibrinogen, collagen type IV,fibronectin, vitronectin, laminin, CCL5 (RANTES), S1PR1, SDF-1, andFGF-4, gels, such as agarose, alginate, and matrigel or solutions suchas PBS, HBS, DMEM EGM or other media. Alternatively, ECM proteins may bepatterned directly onto glass surfaces or porous membranes prior toadhesion of biochips to surface slides using proteinmicro/nano-stamping, or following microfluidic device assembly usingparallel microfluidic streams. Local component concentration may beadjusted by regulating the microfluidic stream flow rate duringinfusion, with focus on alignment and 3-D arrangement.

In other implementations, recapitulating human BM vasculature may beachieved by selectively coating the second channel 104 by culturing withendothelial cells at 37 degrees Celsius and 5 percent CO₂. Endothelialcells may be fixed with 4% formaldehyde, and probed for cellularbiomarkers to resolve cellular localization and architecture. The secondchannel 104 of endothelialized BM biochips may be perfused with afluorescent or colorimetric medium such as FITC-dextran or with beads,and visualized by live-cell microscopy to assess sample/cell/moleculediffusion and determine vascular permeability.

Turning now to FIG. 9, another non-limiting example of a biomimeticsystem 900 is shown, in accordance with various embodiments of thepresent disclosure. The system 900 includes a substrate 902, wherein afirst, or central channel 904, along with a second and third channel, orside channels 906, lateral or adjacent to the central channel 904, areformed. Each channel of system 900 may be configured to carry a flow ofany fluid medium transporting or consisting of but not limited to, forexample, particles, cells, substances, particulates, materials,compositions, agents and the like. In some aspects, system 900 and/orthe substrate 902 may be constructed using cell-inert silicon-basedorganic polymers, such as polydimethylsiloxane (PDMS), COP, COC, PC, PS,PMMA, glass, and/or any other suitable materials or combinationsthereof. In certain configurations, the substrate 902 may include, or beassembled from, separable, re-sealable, or bondable components, such asa first portion 903 and second portion 905 of the substrate, fashionedand/or combined using appropriate techniques and methods.

The central channel 904 and side channels 906 extend substantiallyparallel along a longitudinal direction 908. In some preferred aspects,the dimensions defining the channels along the longitudinal direction908 and the transverse directions, 910 and 912, may be in a rangeconsistent with physiological structures. Moreover, certain dimensionsmay also be desirable to facilitate deposition of cells, substances,compositions or other materials within the channels, or to sustain,regulate or reproduce desired fluid flow profiles, velocities,pressures, or rates, such as those associated with physiologicalsystems. In some designs, dimensions of the central channel 904 and sidechannels 906 along transverse directions 910 and 912 need not be equal,as illustrated in FIG. 9. By way of example, a longitudinal dimension914 of all channels may be in the range of 1000 to 30,000 micrometers,while transverse dimensions for each channel may be in the range of 10to 3,000 micrometers or, more particularly, in the range of 10 to 150micrometers. Other values for the longitudinal and transverse dimensionsare also possible.

The channels are separated by columns 916, generally arranged parallelto the longitudinal direction 908, and extending for a distancesubstantially equal to the longitudinal dimension 914 of the channels.The columns 916 may include any number of apertures 918, that create apartial fluid communication path between the channels, the apertures 918being shaped, dimensioned and arranged, as desired. The apertures 918may be, for example, openings, slits, pores, gaps, microchannels, andthe like, that extend between the central channel 904 and each of firstand/or second side channels 906. As an example, the longitudinal andtransverse dimensions of the apertures 918 may be in the range of 0.1 to20 micrometers, although other values are possible. As illustrated inFIG. 9, the apertures 918 may have a greater longitudinal dimension thantransverse dimensions. In some preferred designs, the apertures 918 aregenerally located proximal to the first portion 903 of the substrate, asillustrated in FIG. 9. To this end, the apertures 918 may extend againstthe first portion 903 of the substrate. This may allow trapped MKsand/or proPLTs, for example, to be pressed against the surface of thefirst portion 903 of the substrate by way of fluid pressure or pressuredifferential generated by traversing fluid medium(s). In the case thatthe first portion 903 of the substrate is transparent, improvedresolution may be achieved with respect to imaging proPLT and PLTproduction processes. In this manner, the apertures 918 may be arranged,shaped and dimensioned to optimally produce, or maximize yield ofdesirable biological substances, such as PLTs.

As shown in FIG. 9, the central channel 904 includes a first channelinput, or central channel input 920, and a first channel output, orcentral channel output 922. The side channels 906 may share a secondchannel input, or side channel input 924, and include separate secondand third channel outputs, or side channel outputs 926. Proximal to theside channel input 924, each side channel 906 of system 900 includes anexpansion portion, or port 928. In certain modes of operation, theexpansion portions, or ports 928 may function as additional fluid inputsor outputs for each of the respective side channel 906, providingincreased flexibility for system 900, as will be described.

The system 900 may also include filtration capabilities, which can takeany shape or form, and are configured to capture or absorb any kind ofdebris, dust and any other contaminants from traversing fluids, whileallowing flow of cells, agents or other desired materials containedtherein, such as MKs. In the non-limiting configuration shown in FIG. 9,filtration elements 930 may be situated in proximity to the side channelinput 924. In addition, the system 900 may also include flow controlcapabilities, which may take a variety of shapes or forms, and designedto control flow forces or damp fluctuations in flow rates. In thenon-limiting configuration shown in FIG. 9, flow resistive elements 932may be situated proximal to channel inputs 920 and 924.

As described with reference to FIG. 1, the channels, columns 916 andapertures 918 may be prepared such that particles, cells, substances,particulates, materials, compositions, and the like, may bind, adhere toor otherwise be confined to any area generally within the channels or inthe vicinity of the columns 916 and apertures 918, and, thereby,allowing harvest of any desired or target biological substance from anarea proximate to the apertures 918. Non-limiting examples ofcompositions, materials or agents, for pre-coating the channels and/orcolumns 916 or and apertures 918 can include, but are not limited to,bovine serum albumin, fibrinogen, fibrinectin, laminin, collagen typeIV, collagen type I, Poly-L-lysine, vitronectin, CCL5, S1PR1, SDF-1,FGF-4, and other extracellular matrix proteins or proteins that regulateplatelet production. In some aspects, such coatings may be performedduring system 900 fabrication steps, or by subsequent perfusion viafluid medium flow through the channels. In addition, the channels may beseeded with cells or other biological compositions and materials, thatinclude, but are not limited to, human or non-human endothelial cells,mesenchymal cells, osteoblasts and fibroblasts. In particular, toreplicate or mimic three-dimensional extracellular matrix organizationand physiological bone marrow stiffness, cells may be infused in ahydrogel solution, which may subsequently be polymerized. The hydrogelsolution may include, but is not limited to, alginate, matrigel, andagarose, which may then be selectively embedded in any channels, asdesired.

Turning to FIG. 10, an example mode of operation for system 900 isshown, depicting an example process 1000 of generating desired or targetbiological substances, such as blood PLTs. Specifically, a firstbiological material or composition is provided via the central channelinput 920 at step 1002. Such biological material may include cellscontained therein for generating PLTs, such as MKs. Then, at step 1004,at least some portions of the biological material may adhere to regionsgenerally about the columns 916 and apertures 918, as described. Forexample, MKs may become trapped in proximity to the apertures 918. Atstep 1006 a target biological substance may be generated by virtue offlow rates, velocities, shear rates, or pressure differentials in thechannels. For example, proPLTs extended as MK's traverse, are trappedabout, or filter through the apertures 918, and subsequently transforminto PLTs. At step 1008 the target biological substance produced iscarried by the fluid medium and may be collected and separated from theeffluent for subsequent use. At step 1010, post-collection processingmay be performed. For example, step 1010 may include a process todialyze the bioreactor-derived platelets in an FDA-approved storagemedia, such as platelet additive solution, such as produced byHaemonetics, COBE Spectra, Trima Accel, and the like. For example, adynamic dialysis system may be used, for example, one that usescontinuous flow at low shear through 0.75 mm, 0.65μ mPES lumen, such asis made by Spectrum Labs. Furthermore, the post-collection processing atstep 1010 may include a process to irradiate the platelet product beforehuman infusion, as required by the FDA. Thus, the culture media may bereplaced with a media that can be infused into human patients.

A unique design feature of system 900 is that media may be selectivelyinfused, and in a bi-directional manner, by virtue of the describedinputs, outputs, and expansion portions, or ports 928 functioning asadditional fluid inputs or outputs. That is, media, cells, or anymaterials, compositions or substances may be separately or concurrentlyintroduced into any or all the channels by both forward or reverse fluidflow. Therefore, in addition to increased harvest efficiency of PLTs,for example, on account of the two side channels 906, the system 900also allows for controlling each channel independently, facilitating ahead-to-head comparison of different operational conditions, such as,media, cells, coating agents, materials, shear rates, fluid flowdirections and so forth. FIG. 11A and 11B illustrate example fluid flowimplementations for producing PLT's, whereby MK's can be introduced intoeither the central channel 904 (FIG. 11A), or each, or both, of the sidechannels 906 (FIG. 11B).

Turning now to FIG. 12, another non-limiting example of a biomimeticsystem 1200 in accordance with various embodiments of the presentdisclosure is shown. The system 1200 includes a substrate 1202. By wayof example, the substrate 1202 may be constructed using cell-inertsilicon-based organic polymers, such as polydimethylsiloxane (PDMS),COP, COC, PC, PS, PMMA, glass, and/or any other suitable materials orcombinations thereof. As shown in FIG. 12, the substrate 1202 includes afirst chamber 1204 and a second chamber 1206 formed therein, wherechambers are separated by a porous membrane 1208.

In some applications, system 1200 may require assembly or disassembly,for example, for individually preparing the first chamber 1204, thesecond chamber 1206, or the porous membrane 1208. As such, system 1200may include a second substrate (not shown in FIG. 12), where the firstchamber 1204 is formed in the first substrate 1202, and the secondchamber 1206 is formed in the second substrate. As such, system 1200 mayinclude additional features that allow the first chamber 1204 and thesecond 1206 to be removably coupled. For example, system 1200 mayinclude components or elements that facilitate breaking or restoring afluid seal between the first chamber 1204, the second chamber 1206, andthe porous membrane 1208.

The first chamber 1204 and the second chamber 1206 and may be shaped anddimensioned in any desired manner, with longitudinal, and transversedimensions selected in dependence of a desired application. Forinstance, in order to produce a target biological substance with adesired yield, such as PLTs, the dimensions of the chambers may beoptimized to control flow rates, velocity profiles, shear rates, shearstresses or pressure differentials between the chambers. As shown in theexample of FIG. 12, the first chamber 1204 and second chamber 1206 mayextend substantially parallel along a longitudinal direction. By way ofexample, the chambers may include longitudinal dimensions in a rangebetween 1000 and 30,000 micrometers, and transverse dimensions in arange between 10 and 300 micrometers, although other values may bepossible.

In some configurations, openings may also be incorporated into a topsurface of first chamber 1204 to permit gas transfer into and out of thesystem 1200. As such, a hydrophobic gas-permeable membrane may belayered proximate to the first chamber 1204 to prevent materials fromescaping through the openings. In such implementation, the system 1200then includes the hydrophobic gas-permeable membrane, the first chamber1204, the porous membrane 1208, and the second chamber 1206, optionallyclamped together using, for example, a re-sealable chip holder, witheach chamber formed in separate substrates, as described.

The porous membrane 1208 may be any element that can create a partialfluid communication path between the first chamber 1204 and secondchamber 1206. For instance, the porous membrane 1208 may be a mesh orfilm having pores, apertures or microchannels configured therein. Theporous membrane 1208 may be fashioned, shaped and dimensioned inaccordance with a particular application. By way of example, the porousmembrane 1208 may be constructed using materials, such as polycarbonatematerials, PDMS, COP, COC, PC, PS, or PMMA. In addition, the porousmembrane 1208 may include lateral and transverse, dimensions in a rangebetween 1 and 100 millimeters, and have a thickness in a range between0.1 to 30 micrometers, and more specifically between 8 and 12micrometers, although other dimensions may be possible. In some designs,the porous membrane 1208 may extend beyond the dimensions of theindividual chambers of system 1200. Specifically, the porous membrane1208 may be configured with a large surface area for trapping desiredbiological materials, cells, and so forth, with capabilities ofsupporting multiple simultaneous proPLT production processes, asdescribed below, hence contributing to increased PLT yield.

Selection of appropriate pore diameter for the porous membrane 1208 maybe such that maximal trapping of cells, such as MKs, may be achieved.For instance, the porous membrane 1208 can have pores in the rangebetween 0.1 to 20 micrometers, and more specifically between 5 and 8micrometers, in diameter. In some aspects, the porous membrane 1208 maybe prepared to include particular materials, chemicals or agents. Forexample, the porous membrane 1208 may include peptides or proteins thatregulate platelet production, such as Poly-L-lysine, fribrinogen,collagen type IV, fibronectin, vitronectin, laminin, CCL5 (RANTES),S1PR1, SDF-1, FGF-4, and so forth.

As shown in FIG. 12, the first chamber 1204 includes an inlet 1210 andoutlet 1212, and similarly the second chamber 1206 includes an inlet1214 and an outlet 1216. In some aspects, the chambers can beindividually prepared using various chemicals, agents or materials byinfusion through respective chamber inlets, using one or more sources,or by incubating the individual layers in the relevant substrate(s). Inother aspects, preparation by infusion of the first chamber 1204 and thesecond chamber 1206 may be performed in parallel. That is, fluidcontaining chemicals, agents or materials may be introduced through bothinlets and collected from both outlets, so that laminar flow streams donot mix.

Non-limiting examples of chemicals, agents, or materials for use withsystem 1200 can include bovine serum albumin (for example, 1-10%),fibrinogen, fibronectin, laminin, collagen type IV, collagen type I, andother extracellular matrix proteins or proteins that regulate plateletproduction. In addition, one or both chambers can be seeded with cellsto recapitulate bone marrow and blood vessel composition by perfusingthem through or incubating the individual layers in the relevant cellculture. These include, but are not limited to, human or mouseendothelial cells, mesenchymal cells, osteoblasts and fibroblasts, andso on. Furthermore, to model three-dimensional extracellular matrixorganization and physiological bone marrow stiffness, cells can beinfused in a hydrogel solution that includes, but is not limited to,alginate, and agarose, that may be polymerized within the system 1200.

System 1200 may also include filtration capabilities, which can take anyshape or form, and be configured to capture or absorb any kind ofdebris, dust and any other contaminants from traversing fluids. In someaspects, filters allow flow of cells, agents or other desired materials,such as MKs, therethrough In addition, the system 1200 may also includeflow control capabilities, which may take a variety of shapes or forms,and be designed to control flow forces or damp fluctuations in flowrates. Furthermore, system 1200 may also include any system, device,source or apparatus configured to establish, sustain, or drain flow ofany medium flowing through system 1200. As described, this can includeone or more sources capable of duplicating physiological conditions byintroducing in the chambers of system 1200 biological compositions atflow rates capable of generating physiological shears between thechambers in a predetermined range, wherein the predetermined range canbe between 100 s⁻¹ and 10,000 s⁻¹.

As shown in FIG. 12, in some designs, multiple copies of system 1200 maybe assembled in an array to produce a biomimetic device or system, witheach copy of system 1200 operating either independently or linked toother copies. Such approach may provide a convenient and efficient wayto parallelize the PLT production process. For instance, an inlet and/oroutlet of one system 1200 may be connected to an inlet and/or outlet ofa second system 1200, and so on. In some designs, inlets may be designedin such a way that biological materials, such as MKs, may be introducedand distributed randomly, or concurrently, into each system 1200 usingone or more sources. In addition, outlets from each system 1200 may beconnected into a single major channel that allows the collection into asingle container of effluent containing a target biological substance,such as proPLTs and PLT's, from every system 1200 in the array.

By way of example, for a porous membrane 1208 of dimensions 50millimeters by 75 millimeters, over 160 systems 1200 may be combinedonto a single biomimetic device. Considering the number of pores foreach system 1200 to be around of 1.2×10⁸, this implies that each devicemay be capable of capturing roughly 2×10¹⁰ cells, which represents avalue high enough to produce sufficient numbers of PLTs for in vivo(animal and human) testing and infusion.

System 1200 includes several advantages, included the capability forspecifically coating a first surface of pores located on the porousmembrane 1208 with defined ECM proteins and a second surface of thepores without these proteins, or with other ECM proteins, ensuring thatMKs may come to rest on the first surface contact their proteins ofinterest, while the proPLTs extended and the PLTs they release contactanother. In addition, the design of system 1200, as described,facilitates cleaning, or swapping of various porous membranes 1208configured from different materials and having different pore sizes, asneeded for a particular applications.

Systems 100, 900, and 1200, in accordance with aspects of the presentdisclosure, provide platforms that replicate or reproduce physiologicalconditions found in human physiology by duplicating dimensions,environments and conditions therein. For instance, microfluidic channelsseparated by columns spaced closely apart, experiencing controlledenvironments and flow conditions, as described with reference to FIG. 1,provide a realistic physiological model that replicates human BM. Bycontrolling MK trapping, BM stiffness, ECM composition, micro-channelsize, hemodynamic vascular shear, and endothelial cell contacts, usingsystems 100, 900, and 1200, functional PLTs may be produced.

Turning to FIG. 13, steps of a process 1300 for producing aphysiological model are shown, where the model can include at least oneof a bone marrow and blood vessel structure. At process block 1302, anynumber of biological compositions may be introduced into a providedbiomimetic microfluidic system as described, for example, with referenceto FIGS. 1, 9 and 12. In some implementations, process block 1302 mayinclude introducing a first biological composition into a first channelor chamber of the provided system at a first flow rate using a firstsource, and introducing a second biological composition into a secondchannel or chamber of the provided system at a second flow rate using asecond source. In other implementations, the second or a thirdbiological composition may be introduced into a third channel or chamberof the system, using the second or a third source at a third flow rate.As described, each channel or chamber may be prepared, processed and/orinfused with biological compositions using any combination of sourcesand flows, where each biological composition can include semi-solids,solids, liquids, cells, and so forth, or a combination thereof.

At process block 1304, flow rates may be controlled in order to createdesired differentials between channels or chambers. In some aspects,controlling such flow rates may generate physiological shear rateswithin a predetermined range that would facilitate production bloodplatelets. For example, such predetermined range may be between 100 s⁻¹and 10,000 s⁻¹, and more specifically between 500 s⁻¹ and 2500 s⁻¹. Insome aspects, respective directions of flow rates may be reversed, asdescribed with reference to FIG. 11. Then, at process block 1306, targetbiological substances produced, for example, proximate to themicrochannels configured in the provided biomimetic microfluidic system,may be harvested from the effluent. As described, such target biologicalsubstances can include blood platelets. In some aspects, the effluentmay undergo a number of processing steps at process block 1306 in orderto extract the target biological substances from the effluent.

Further examples of materials and methods utilized in these approachesare detailed below. It will be appreciated that the examples are offeredfor illustrative purposes only, and are not intended to limit the scopeof the present disclosure in any way. Indeed, various modifications inaddition to those shown and described herein, such as applicability tothe blood brain barrier or molecular diffusion across separate mediums,may be possible. For example, specific implementations, includingdimensions, configurations, materials, cell types, particulates, flowmedium and flow rates, fabrication methods and recipes, as well asimaging, processing and analysis methods, and so on, are described.However, it will be appreciated that implementations may also be used,and still fall within the scope of the appended claims.

EXAMPLES Microfluidic Device Design and Fabrication

Microfluidic devices were fabricated using soft lithography. As shown inthe example of FIG. 1, the devices included two channels containingpassive filters, for trapping air bubbles and dust, followed by fluidresistors, used to damp fluctuations in flow rate arising duringoperation. The channels merged to a rectangular area 1300 micrometerslong, 130 micrometers wide, and 30 micrometers deep, separated by aseries of columns (10 micrometers wide and 90 micrometers long) spaced 3micrometers apart. To ensure efficient gas exchange and supporthigh-resolution live-cell microscopy during cell culture, themicrofluidic devices were constructed from a cell-inert silicon-basedorganic polymer bonded to glass slides.

AutoDesk software in AutoCAD was used to design the desired 2D patternand printed on a photolithography chrome mask. Silicon wafers(University Wafers, Boston, Mass.) were spin coated with SU-8 3025photoresist (Michrochem, Newton, Mass.) to a 30 micrometers filmthickness (Laurel) Technologies, North Wales, Pa.), baked at 65 degreesC. for 1 minute and 95 degrees C. for 5 minutes, and exposed to UV light(˜10 mJ cm⁻²) through the chrome mask for 30 seconds. The unbound SU-8photoresist was removed by submerging the substrate into propyleneglycol monomethyl ether acetate for 7 minutes. Polydimethylsiloxane(PDMS) was poured onto the patterned side of the silicon wafer,degassed, and cross-linked at 65 degrees C. for ˜12 hours. After curing,the PDMS layer was peeled off the mold and the inlet and outlet holeswere punched with a 0.75 mm diameter biopsy punch. The channels weresealed by bonding the PDMS slab to a glass cover slide (#1.5, 0.17×22×50mm, Dow Corning, Seneffe, Belgium) following treatment with oxygenplasma (PlasmaPrep 2, GaLa Instrumente GmbH, Bad Schwalbach, Germany).Samples were infused into the microfluidic device via PE/2 tubing(Scientific Commodities, Lake Havasu City, Ariz.) using 1 mL syringesequipped with 27-gauge needles (Beckton Dickinson, Franklin Lakes,N.J.). Flow rates of liquids were controlled by syringe pumps (PHD 2000,Harvard Apparatus, Holliston, Mass.).

Microfluidic Device Operation

Devices were coated with a 0.22 μm filtered 10% BSA solution (Millipore,Billerica, Mass.) for 30 minutes to prevent direct cell contact withglass. Referring to FIG. 1, primary MKs and media were infused in thefirst inlet 118 and second inlet 122, respectively, at a rate of 12.5μL/hour using a two-syringe microfluidic pump (Harvard Apparatus,Holliston, Mass.). When the first outlet 120 was closed, both inputsolutions were redirected toward the second outlet 124 causing primaryMKs to trap.

Extracellular Matrix Composition Modeling (2D)

Microfluidic devices were selectively coated with extracellular matrixproteins by perfusing the channels with rhodamine-conjugated fibrinogen(1 mg/mL) or fibronectin (50 μg/mL, Cytoskeleon Inc., Denver, Colo.) for30 minutes. Samples were perfused in parallel through both inlets andcollected through both outlets so that laminar flow streams did not mix.Devices were washed with 1×PBS and coated with 0.22 μm filtered(Millipore, Billerica, Mass.) 10% BSA solution (Roche, South SanFrancisco, Calif.) for 30 minutes to coat any remaining exposed glass.

BM Stiffness Modeling (3D)

Primary MKs were re-suspended in 1% sterile alginate with an averagemolecular weight of 150-250 kD (Pronova SLG100, FMC biopolymer, Norway)in culture media and perfused across the microfluidic device (firstinlet 118, second outlet 124 in FIG. 1) until MKs became trapped. Thesecond channel was then selectively perfused with 1×PBS to removealginate from this channel. To make a homogenous alginate gel, 30 mMnanoparticle calcium carbonate (mkNANO, Canada) was used as a calciumsource and dissolved in 60 mM slowly hydrolyzing D-Glucono-5-lactone(Sigma-Aldrich, St. Louis, Mo.), which releases the calcium in thesolution (in review Khavari et al NJP 2013). The calcium carbonatesuspension was perfused along the second channel until the alginatesolution retained in the first channel became polymerized (˜20 minutes).The second channel was then selectively washed with 1×PBS and replacedwith culture media. To determine the alginate gel's mechanicalproperties 0.25 percent, 0.5 percent, 1.0 percent, and 2.0 percentalginate gels were prepared and their frequency-dependent shear moduliwere measured by rheology at 37° C. (Ares G2 TA instruments, New Castle,Del.).

Sinusoidal Blood Vessel Contact Modeling (3D)

Microfluidic devices were selectively coated with 50 μg/mL fibronectin(Cytoskeleon Inc., Denver Colo.) and 10 percent BSA (Roche, South SanFrancisco, Calif.), as described above, and transferred to a 37 degreesC., 5 percent CO2 incubator. 10,000,000 HUVECs/mL in EBM media (Lonza,Basel, Switzerland) were seeded over the fibronectin-coated channel at12.5 uL/hour and permitted to adhere to this surface over a period of 3hours. The inlet sample was replaced with cell-free EBM media andperfused through the channel until HUVECs reached confluency (2-8 days).Cells were stained with 5 μM CellTracker Red and 1 μg/mL Hoescht 33342(Invitrogen, Carlsbad, Calif.) for 45 minutes, washed in fresh media orfixed in 4% formaldehyde and visualized by confocal-fluorescencemicroscopy.

Vascular Shear Rate Modeling (3D)

The shear stresses imparted on the MKs were estimated with acomputational model of the fluid dynamics within the microfluidicdevice. A commercial finite element method software (COMSOL) was used tosolve the Navier-Stokes equation. The steady-state Navier Stokes flowequation for incompressible flow is:

ρ(

)=−

p=μ

+f  (1)

where ρ is the fluid density, ν is the flow velocity, p is the pressure,μ is the fluid viscosity and f is the body forces action on a fluid.Equation (1) was solved in a three dimensional computational domainreplicating the exact dimensions of the microfluidic device. It wasassumed that the fluid within the device had a viscosity and density ofwater (0.001 Pa s and 1000 kg/m3, respectively). No slip boundaryconditions were assumed at the walls of the channels. The infusion flowrates ranged from 12.5-200 μL/hr. A triangular mesh, which was madefiner at the slits, was used to discretize the domain. The modelcontained 315,317 degrees of freedom. Mesh independence, as wasconfirmed by obtaining less than a 10 percent difference between shearrates, was found between 251,101 and 415,309 degrees of freedom. Thesteady state solutions were obtained using the UMFPACK linear systemsolver.

Primary Mouse Megakaryocyte Culture

Mouse FLCs were collected from WT CD1 mice (Charles River Laboratories,Wilmington, Mass.) and MKs were cultured.

Electron Microscopy

Megakaryocyte input and bioreactor effluent were fixed with 1.25 percentparaformaldehyde, 0.03 percent picric acid, 2.5 percent glutaraldehydein 0.1-M cacodylate buffer (pH 7.4) for 1 h, post-fixed with 1% osmiumtetroxide, dehydrated through a series of alcohols, infiltrated withpropylene oxide, and embedded in epoxy resin. Ultrathin sections werestained and examined with a Tecnai G2 Spirit BioTwin electron microscope(Hillsboro, Oreg.) at an accelerating voltage of 80 kV. Images wererecorded with an Advanced Microscopy Techniques (AMT) 2-K chargedcoupled device camera, using AMT digital acquisition and analysissoftware (Advanced Microscopy Techniques, Danvers, Mass.).

Immunofluorescence Microscopy

Megakaryocytes, released proPLTs, or bioreactor effluent were purifiedand probed. Samples were either incubated with 5 μM CellTracker Green(Invitrogen, Carlsbad, Calif.) for 45 minutes, washed in fresh media andvisualized by live-cell fluorescence microscopy, or fixed in 4%formaldehyde and centrifuged onto poly-L-lysine (1 μg/mL)-coatedcoverslides. For analysis of cytoskeletal components, samples werepermeabilized with 0.5 percent Triton-X-100, and blocked inimmunofluorescence blocking buffer (0.5 g BSA, 0.25 ml 10% sodium azide,5 ml FCS, in 50 ml 1×PBS) overnight before antibody labeling(55). Todelineate the microtubule cytoskeleton, samples were incubated with arabbit polyclonal primary antibody for mouse or human β1-tubulin. Todelineate the actin cytoskeleton, samples were incubated with Alexa 568phalloidin (Invitrogen, Carlsbad, Calif.). Cell nuclei were labeled with1 μg/mL Hoescht 33342 (Invitrogen, Carlsbad, Calif.). To correct forbackground fluorescence and nonspecific antibody labeling, slides wereincubated with the secondary antibody alone, and all images wereadjusted accordingly. Samples were examined with a Zeiss Axiovert 200(Carl Zeiss, Thornwood, N.Y.) equipped with 10× (numerical aperature,0.30) Plan-Neofluar air and 63× (numerical aperature, 1.4)Plan-ApoChromat oil immersion objectives, and images were obtained usinga CCD camera (Hamamatsu Photonics, Boston, Mass.). Images were analysedusing the Metamorph version 7.7.2.0 image analysis software (MolecularDevices, Sunnyvale, Calif., USA) and ImageJ version 1.47p software (NIH,http://rsb.info.nih.gov.ezp-prod1.hul.harvard.edu/ij/).

Cell Size and Morphology Determination

Cells were individually thresholded and high-content cytoplasmic areaand perimeter measurements were performed in ImageJ usinginvestigator-coded software, outlined below. Analysis was confirmed bymanual inspection of all samples, and improperly thresholded cells wereexcluded from the analysis. MK diameters were calculated from areameasurements to account for non-circular cells. More than 2000 cellswere counted for each condition, and analysis of MK area and effluentcomposition was performed for at least three independent samples.Statistical significance was established using a 2-tailed Student t testfor paired samples. Error bars represent one standard deviation aboutthe mean.

Live Cell Microscopy

For shear cultures, MKs were loaded onto ‘naked’ microfluidic devices(only BSA-coated), and the infusion rate was doubled incrementally from12.5 μL/hr to 200 μL/hr over a 2 hour period. For static cultures,isolated MKs were pipetted into chambers formed by mounting a glasscoverslide coated with 3% BSA onto a 10 mm petri dish with a 1 cm holeand cultured for 24 hours. Both static and shear cultures weremaintained at 37 degrees C. and 5 percent CO2 and examined on a ZeissAxiovert 200 (Carl Zeiss, Thornwood, N.Y.) equipped with 10× (numericalaperature, 0.30) Plan-Neofluar air objective. Differential interferencecontrast (DIC) images were obtained using CCD camera (HamamatsuPhotonics, Boston, Mass.) at either 2 second (shear cultures) or 20minute (static cultures) intervals. Images were analyzed using theMetamorph version 7.7.2.0 image analysis software (Molecular Devices,Sunnyvale, Calif., USA) and ImageJ software version 1.47p. ProPLTextension rates were determined manually for over 200 MKs from at leastthree independent samples. For PLT spreading experiments, effluent wascollected from microfluidic devices after 2 hours and pipetted intouncoated static culture chambers, described above. PLTs were permittedto contact glass by gravity sedimentation and spreading was captured at5 second intervals over a 5 minute period.

GFP-β1 Tubulin Retro Viral Transfection

Dendra2-fused β1 tubulin was cloned into pMSCV plasmids. HEK 293 cellspackaging cells were cultured in DMEM supplemented with 10% fetal bovineserum (FBS) to 30-50 percent confluency. Transfection of HEK 293 cellswas performed using 2 μg of DNA plasmids encoding gag/pol, vsvG, and the⊕1 tubulin fused with Dendra2 in the pMSCV vector. After medium exchangethe following day, cells were incubated for 72 hours for virusproduction. The supernatant was filtered through a 0.22 μm filter(Millipore, Billerica, Mass.), and aliquots were stored at −80 degreesC. On the second day of culture, MKs isolated from fetal liver culturesdescribed above were resuspended in DMEM containing 10 percent FBS, 8μg/mL polybrene (Sigma), and the retroviral supernatant. Samples weretransferred to a 6-well plate, centrifuged at 800×g for 90 minutes at 25degrees C. and then incubated at 37 degrees C. for 90 minutes. Followingincubation, MKs were washed by centrifugation and resuspended in freshDMEM containing 10 percent FBS and TPO. MKs were allowed to mature untilday 4 of culture and then isolated by a BSA gradient, as previouslydescribed.

Flow Cytometry

Platelets were collected from the released proPLT fraction of static MKcultures or bioreactor effluent and examined under resting conditions.Samples were probed with FITC-conjugated antibodies against CD42a orCD41/61 (Emfret Analytics, Eibelstadt, Germany) and run on a FACSCaliburflow cytometer (Beckton Dickinson). PLTs were gated by theircharacteristic forward- and side-scattering as they passed through thedetector, and their total fluorescence intensity was calculated aftersubtraction of a FITC-conjugated IgG antibody specificity control(Emfret Analytics). Quantization of PLT yield was determined by dividingnet GP IX+PLT production by net GP 1X+MK depletion over effluentcollection period, and was performed for at least 3 independent samplesResults were identical for GP IIbIIIa+cells.

Image Analysis

The digital images acquired in Metamorph were analyzed using ImageJ andAdobe Photoshop CS3 (Adobe Systems, San Jose, Calif.). Dividing linesexplicitly separate different images, or separate regions of the sameimage. No specific features within an image were enhanced, obscured,moved, removed, or introduced, and adjustments made to the brightness,contrast, and color balance were linearly applied to the whole image.

Microfluidic Device Models Physiological Characteristics of Human BM

To recapitulate physiological conditions, each of the channels wereselectively coated with fibrinogen and fibronectin, respectively toreproduce ECM composition of the BM and blood vessel microenvironments(shown in FIG. 2A). By running flow across the microfluidic device,primary MKs infused along a first channel would become sequentiallytrapped between the columns and extend proPLTs into the second channel(shown in FIG. 2B), recapitulating physiological proPLT extension. Tomodel 3D ECM organization and physiological BM stiffness (250 Pa), MKswere infused in a 1 percent alginate solution that was polymerizedwithin the microfluidic device, selectively embedding the MKs inalginate gel within the first channel while retaining vascular flow inthe second channel. Alginate did not inhibit proPLT production, and MKdistance from the second channel could be controlled.

Human umbilical vein endothelial cells (HUVECs) were selectively seededalong the second channel, and grown to confluency to produce afunctional blood vessel (shown in FIG. 2C). In addition, MK behavior wasmonitored by 10×-150× magnification, high-resolution live-cellmicroscopy, and the released PLTs were collected from the effluent. FIG.2D shows the complete system illustrating operation. Laminar fluid shearrates were characterized (shown in FIG. 2E), and were tightly controlledusing two microfluidic pumps (one for the first channel and one for thesecond channel). Shear rates within the device were linearlyproportional to infusion rates and were adjusted to span thephysiological range (500-2500 s⁻¹). While shear rates at emptymicrochannel junctions increased with distance from the first channel(shown in FIG. 2F), upon a MK trapping, flow was redirected to the nextavailable gap such that MKs continued to experience physiological(between 760 and 780 s⁻¹) shear rates at these sites (shown in FIG. 2G).

Vascular Shear Triggers ProPLT Production, Physiological Extension, andRelease

In vivo BM MKs extend proPLTs in the direction of blood flow and releasePLTs, proPLTs, large cytoplasmic fragments (prePLTs), and even whole MKsinto sinusoidal blood vessels which may be trapping in the pulmonarymicrovascular bed, or otherwise maturing in the circulation. Todetermine the effect of physiological shear on PLT production, mousefetal liver culture-derived (mFLC) MKs were isolated on culture day 4and characterized by size and ploidy before being infused into themicrofluidic device (shown in FIG. 3A).

One of the major challenges in producing transfuseable PLTs in vitro hasbeen identifying factors that trigger proPLT production. Under staticconditions MKs begin producing proPLTs ˜6 hours post-isolation, andreach maximal proPLT production at 18 hours (shown in FIG. 3B). Bycomparison, MKs under physiological shear (shown in FIG. 3C at roughly500 s⁻¹) began producing proPLTs within seconds of trapping, reachingmaximal proPLT production and biochip saturation within the first 2hours of culture. MKs cultured under physiological shear produced fewer,longer proPLTs that were less highly branched relative to staticcultures. ProPLTs in shear cultures were uniformly extended into thelower channel and aligned in the direction of flow against the vascularchannel wall, recapitulating physiological proPLT production. Thepercent of proPLT-producing MKs under physiological shear were doubledover static cultures to roughly 90% (shown in FIG. 3D).

Another major challenge in generating clinical numbers of PLTs forinfusion has been that in vitro cultures extend proPLTs at asignificantly slower rate than what has been observed in vivo.Application of physiological shear in our microfluidic device increasedproPLT extension rate by an order of magnitude above static culturecontrols to roughly 30 μm/min (shown in FIG. 3E), which agrees withphysiological estimates of proPLT extension rate from intravitalmicroscopy studies in living mice and support increased PLT productionin vitro.

Early histological studies in both humans and mice have predicted thatwhole MKs, as well as MK fragments may be squeezing through gaps orfenestrations in the vascular endothelium lining BM blood vessels totrap in the pulmonary circulatory bed. Large PLT intermediates calledprePLTs were recently discovered in blood, and venous infusion of mBM-and FLC-derived MKs and prePLTs into mice produced PLTs in vivo. In thepresent study, 100 μm+ diameter MKs were routinely observed squeezingthrough 3 μm (shown in FIG. 4A) and 1.5 μm gaps, or extending large MKfragments (shown in FIG. 4B), supporting a model of vascular PLTproduction. In addition, abscission events were routinely captured byhigh-resolution live-cell microscopy and occurred at variable positionsalong the proPLT shaft, releasing both prePLT-sized intermediates (3-10μm diameter) and PLTs (1.5-3 μm diameter) (shown in FIG. 4C and FIG.4D). Following each abscission, the resulting proPLT end formed a newPLT-sized swelling at the tip, which was subsequently extended andreleased, repeating the cycle (shown in FIG. 4E).

While shear rates were kept constant, proPLT extension rates varied atdifferent positions along the shaft, predictive of a regulatedcytoskeletal driven mechanism of proPLT elongation (shown in FIG. 4C).Increasing microfluidic shear rates within the physiological range didnot affect the median proPLT extension rate or the distribution ofproPLT extension rates in culture (shown in FIG. 4F), and proPLTprojections in MKs retrovirally transfected to express GFP-β1 werecomprised of peripheral microtubules (MTs) that formed coils at thePLT-sized ends (shown in FIG. 4G). ProPLTs reached lengths exceeding 5mm, and resisted shear rates up to 1000 s⁻¹ in vitro; recapitulatingphysiological examples of proPLT production from intravital microscopy,and demonstrating that abcission events were not caused by shear. Toconfirm that shear-induced proPLT extension was cytoskeletal-driven, MKswere incubated with 5 μM Jasplankinolide (Jas, actin stabilizer) or 1 mMerythro-9-(3-[2-hydroxynonyl] (EHNA, cytoplasmic dynein inhibitor) priorto infusion in microfluidic device. Both Jas and EHNA inhibitedshear-induced proPLT production (shown in FIG. 4H and FIG. 4I) and PLTrelease under both static and physiological shear conditions.

Derived PLTs Manifest Structural and Functional Properties of Blood PLTs

PLTs are anucleate discoid cells ˜1-3 μm in diameter that expressbiomarkers GP IX and IIbIIIa on their surface, and are characterized bya cortical MT coil of 6-8 MTs encircling an actin-based cytoskeletalnetwork. To establish PLT yield, biomarker expression, and forward/sidescatter and relative concentration of glycoprotein (GP) IX+mFLC-MKs weremeasured by flow cytometry immediately before infusion in ourmicrofluidic device on culture day 4 (shown in FIG. 5A). Effluent wascollected 2 hours post infusion and compared to mFLC-MK input (shown inFIG. 5B). Input MKs and effluent PLTs both expressed GP IX and IIbIIIaon their surface, and displayed characteristic forward/side scatter. Theapplication of shear shifted the cellular composition of the effluenttoward more PLT-sized GPIX+ cells relative to static culture supernatantisolated on culture day 5 (shown in FIG. 5C). 85±1% of MKs wereconverted into PLTs over 2 hours, which agreed with our quantitation ofpercent proPLT production (FIG. 3D) and constitutes a significantimprovement over static cultures (FIG. 5D). Continuous perfusion ofroughly 500 s⁻¹ shear over 2 hours in our microfluidic device yieldedroughly 21 PLTs per MK and constitutes a major advance in PLT productionrate over existing culture approaches that generate comparable PLTnumbers over a much longer period of time (6-8 days).

To quantify the morphological composition of our product, the effluentfrom our microfluidic device was probed for β1 tubulin (PLT-specifictubulin isoform) and Hoescht (nuclear dye), and analyzed byimmunofluorescence microscopy. Cells were binned according to theirmorphology and size, and compared to static MK culture supernatants. Theapplication of shear shifted the cellular composition of the effluenttoward more PLT-sized β1 tubulin+Hoescht− cells (shown in FIG. 5E),which agreed with flow cytometry data (FIG. 5C) and resulted in aproduct that was more similar in composition to the distribution of PLTintermediates in whole blood. Quantitation of free nuclei in effluentconfirmed increased microfluidic device-mediated PLT production relativeto static cultures and established PLT yields of roughly 20±12 PLTs perMK, which agree with flow cytometry data.

Resting PLTs contain characteristic invaginations of the surfacemembrane that form the open canalicular system, a closed channel networkof residual endoplasmic reticulum that form the dense tubular system,organelles, specialized secretory granules, and will flatten/spread oncontact activation with glass. Microfluidic device-generated PLTs wereultrastructurally indistinguishable from mouse blood PLTs bythin-section transmission electron; and contained a cortical MT coil,open canalicular system, dense tubular system, mitochondria, alpha- anddense-granules (as shown in FIG. 5F). Microfluidic device-generated PLTsand PLT intermediates displayed comparable MT and actin organization tomouse blood PLTs by immunofluorescence microscopy (as shown in FIG. 5G),and spread normally on contact-activation with glass, forming bothfilpodia and lamellipodia.

Application of the Microfluidic Device to Human PLT Production

To generate human PLTs, mFLC-MK in our microfluidic device were replacedwith hiPSC-derived MK, which provide a virtually unlimited source of MKsfor infusion. hiPSC-MKs were isolated on culture day 15, once they hadreached maximal diameter of 20-60 μm (shown in FIG. 6A), and wereultrastructurally similar to primary human MKs (shown in FIG. 6B). Instatic culture, hiPSC-MKs began producing proPLTs at 6 hourspost-isolation, and reached maximal proPLT production at 18 hours (shownin FIG. 6C). By comparison, hiPSC-MKs under physiological shear (about500 s^('1)) began producing proPLTs immediately upon trapping, andextended/released proPLTs within the first 2 hours of culture (shown inFIG. 6D). The percent proPLT-producing hiPSC-MKs under shear wereincreased significantly over static cultures (˜10%) to roughly 90% (asshown in FIG. 6E).

ProPLT extension rates were slightly lower than mFLC-MK controls (˜19μm/min versus 30 μm/min) (shown in FIG. 6F) and more closelyapproximated physiological controls. Microfluidic device-generated PLTsdisplayed forward and side scatter, and surface biomarker expressioncharacteristic of human blood PLTs, were ultrastructurallyindistinguishable from human blood PLTs by thin-section transmissionelectron (shown in FIG. 6G), displaying comparable MT and actinexpression to human blood PLTs by immunofluorescence microscopy (shownin FIG. 6H), spreading normally on contact-activation with glass, andforming both filpodia and lamellipodia (shown in FIG. 6I). Takentogether these data demonstrate that hiPSC-MKs can be applied to ourbiomimetic microfluidic device to generate potentially unlimited numbersof functional human PLTs.

Application of the Microfluidic Device to Drug Development

Thrombocytopenia may appear suddenly and often unintentionally,potentially causing major bleeding and death. Antibody and cell-mediatedautoimmune responses have been shown to cause thrombocytopenia. Inaddition, thrombocytopenia may also be triggered by a wide range ofmedications, including cancer drugs, such as dasatinib. Animal modelsare generally poor predictors of safety and efficacy of medications inhumans, and clinical studies are time-consuming, expensive, andpotentially harmful. Microfluidic devices designed to mimic human BMrepresent an area of innovation of major clinical importance, offeringan efficient and realistic platform to investigate the effects of avariety of medications upon BM and MK biology.

PLT survival and clearance rates are usually measured through infusionstudies using flow cytometry. Quantification of the rate and extent ofproPLT production, however, is not amenable to this approach, andrequires direct visualization to establish at what stagethrombocytopoiesis is affected. By contrast, the application ofmicrofluidic systems, in accordance with the present disclosure, offersa great platform to study drug effects on PLT production, one that mayfacilitate the identification of new regulators of PLT production andelucidate the mechanism of clinically significant drug-inducedthrombocytopenias.

As proof of concept, high-content live-cell microscopy was employed toidentify the express GFP.PI tubulin (live-cell microscopy) mechanism bywhich trastuzumab emtansine (T-DM1), an antibodydrug conjugate currentlyin clinical development for breast cancer, affects PL T production.These studies revealed that T-DM1 inhibits MK differentiation, anddisrupts proPLT formation by inducing abnormal tubulin organization (asshown in FIG. 7). Defining the pathways by which therapeutics such asT-DM1 affect MK maturation and proPLT production may yield strategies tomanage drug-induced thrombocytopenias and regulate PLT production invivo.

The approach of the present disclosure capitalizes on a highly novelmicrofluidic design to recapitulate human BM and blood vessel physiologyex vivo, and generate an alternative source of functional human PLTs forinfusion. While clinically desirable to meet growing transfusion needsand obviate risks currently associated with platelet procurement andstorage, 2 major quantitative roadblocks have thus far persisted in thedevelopment of donor-independent PLTs for therapeutic use: (1)generating sufficient numbers (˜3×10⁸) of human MKs to support theproduction of one PLT transfusion unit (˜3×10¹¹ PLTs), and (2)generating physiological numbers of functional human PLTs (˜10³-10⁴) perMK. The development of human embryonic stem cell cultures (hESC), andmore recently, human induced pluripotent stem cell cultures (hiPSC),offer a potentially unlimited source of progenitor cells that can bedifferentiated into human MKs in vitro to address the first quantitativeroadblock. Indeed, because PLTs are anucleate, PLT microfluidicdevice-derived units could be irradiated prior to infusion, addressingconcerns that cellular products derived from hESC or hiPSCs could beoncogenic or teratogenic.

Attempts to study the environmental drivers of PLT production have beenconstrained by reductionist approaches, and a major limitation of 2Dliquid cultures has been their inability to account for 3D BMcomposition and stiffness, directionality of proPLT extension, andproximity to venous endothelium. Likewise, while proPLT-producing MKsentering sinusoidal blood vessels experience wall shear rates of 500 to2500 s⁻¹, attempts to model vascular flow by perfusing MKs overECM-coated glass slides have selected for immobilized/adhered MKs, andhave been unable to discriminate ECM-contact activation from shear.Alternatively, released proPLTs have been centripetally agitated in anincubator shaker, which does not recapitulate laminar shear flow in BMblood vessels, does not provide precise control of vascular shear rates,and is not amenable to high-resolution live-cell microscopy.Nonetheless, despite these limitations, exposure of MKs to high shearrates (1800 s⁻¹) accelerated proPLT production, and proPLTs cultured inthe absence of shear released significantly fewer PLTs than thosemaintained at fluid shear stresses of ˜0.5 Pa for 2 hours. Moreover,recent advances in multiphoton intravital microscopy have providedincreasing resolution of proPLT production in vivo and confirmed theimportance of vascular flow on proPLT extension and PLT release. Whilethese studies have provided physiologically accurate examples of in vivoproPLT production, poor resolution and limited control of themicroenvironment has prohibited detailed study of how the BMmicroenvironment contributes to PLT release.

Mounting evidence that cell-cell contacts, extracellular matrix (ECM)composition and stiffness, vascular shear rates, pO2/pH, soluble factorinteractions, and temperature contribute to proPLT formation and PLTrelease have suggested that recapitulating BM and blood vesselmicroenvironments within a 3D microfluidic culture system is necessaryto achieve clinically significant numbers of functional human PLTs.Indeed, modular 3D knitted polyester scaffolds have been applied undercontinuous fluid flow to produce up to 6×10⁶ PLTs/day from 1×10⁶ CD34+human cord blood cells in culture. While suggestive that clinicallyuseful numbers of culture-derived human PLTs are attainable, 3Dperfusion bioreactors have not accurately reproduced the complexstructure and fluid characteristics of the BM microenvironment, andtheir closed modular design has prevented direct visualization of proPLTproduction, offering little insight into the mechanism of PLT release.Alternatively, 3D PDMS biochips adjacent ECM-coated silk-based tubeshave been proposed to reproduce BM sinusoids and study MKdifferentiation and PLT production in vitro. While capable ofrecapitulating MK migration during maturation, this design is notamenable to high resolution live-cell microscopy, and does not reproduceendothelial cell contacts necessary to drive MK differentiation.

By comparison, the microfluidic device design of the present disclosureoffers the complete package, allowing significant improvement in time toPLT release and an increased total PLT yield. Also, application ofvascular shear rates within the microfluidic device induces proPLTproduction, and reproduces physiological proPLT extension and release.Furthermore, MKs are capable of squeezing through small gaps to enterthe circulation and releasing prePLT intermediates under physiologicalflow conditions. The product resulting from continuous perfusion of MKsin the microfluidic device of the present disclosure approachedphysiological PLT concentrations, and manifested both structural andfunctional properties of blood PLTs. Finally, PLT microfluidic devicescould be applied to human MK cultures to produce functional human PLTs.Although PLT yield per MK fell short of theoretical estimates, theobservation that MK cultures routinely released large MK fragments(prePLTs, proPLTs) as well as MK themselves into the effluent channel,suggests that actual PLT numbers may depend on the furtherdifferentiation of PLT intermediates into PLTs in supportivemicroenvironments such as the lung or circulating blood. Indeed, whenmFLC-derived proPLTs were infused into mice, these were rapidlyconverted into PLTs over a period of 12-24 hours. Interestingly, whileCMFDA-labeled PLTs in this study were readily detected in the blood,larger prePLT intermediates were not, suggesting that they may betrapping in a microcirculation of the lung. Likewise, when mFLC andBM-derived MKs were infused into mice they almost exclusively localizedto the lungs and released PLTs within the first two hours. In bothcases, it is almost certain that vascular shear rates, soluble factorinteractions in the blood, and endothelial cell contacts regulate thisprocess, and examining how local microenvironments in these tissuescontribute to terminal PLT production warrant further investigation.

By combining the major elements of BM physiology including 3D ECMcomposition and stiffness, cell-cell contacts, vascular shear rates,pO2/pH, soluble factor interactions, and temperature within a singlemicrofluidic system, the approach of the present disclosure offersunprecedented control of ex vivo microenvironments and a biomimeticplatform for drug development. Moreover, support of high-resolutionlive-cell microscopy permits direct visualization of cells duringculture and provides a window into poorly characterized physiologicalprocesses. Lastly, the microfluidic device design can be easily scaledby mirroring effluent channels on either side of a central channel,elongating the device to support greater numbers of columns, andpositioning multiple units in parallel within a larger microfluidicdevice matrix. Continuous harvesting of hiPSC-MKs in longer devices mayresult in clinically significant numbers of PLTs to perform, forexample, traditional aggregometry tests of PLT function, and in vivoxeno-transfusion studies in immune-suppressed mice to measure increasesin PLT counts, which require roughly 10⁸ PLTs per study.

In summary, the present disclosure has demonstrated systems and methodsfor reproducing human BM and sinusoidal blood vessel microenvironmentsfor generating human platelets in an approach amenable to highresolution imaging. Biomimetic microfluidic systems, in accordance withthe present disclosure, may be fabricated using PDMS, glass and anyother suitable materials, and include several microfluidic channel andchamber configurations designed to simulate realistic physiologicalconditions, such as flow velocities, shear rates, pressuredifferentials, and so forth. As such, the channels or chambers ofmicrofluidic systems described herein may be selectively coated with ECMand human endothelial cells, as well as other biological agents ormaterials consistent with physiological systems. In some forms ofoperation, as described, round or proPLT-producing MKs, infused alongdifferent channels of the microfluidic systems detailed herein, maysequentially become trapped, and extend platelet-producing proPLTs intoadjacent channels that subsequently release PLTs for harvest. Suchprocesses may be stimulated or optimized by controllable physiologicalshear rates and regulated microenvironments, and the released PLTsentering the fluid stream can be collected from the effluent, with theprocess being capable of visualization using, for example,high-resolution microscopy.

The various configurations presented above are merely examples and arein no way meant to limit the scope of this disclosure. Variations of theconfigurations described herein will be apparent to persons of ordinaryskill in the art, such variations being within the intended scope of thepresent application. In particular, features from one or more of theabove-described configurations may be selected to create alternativeconfigurations comprised of a sub-combination of features that may notbe explicitly described above. In addition, features from one or more ofthe above-described configurations may be selected and combined tocreate alternative configurations comprised of a combination of featureswhich may not be explicitly described above. Features suitable for suchcombinations and sub-combinations would be readily apparent to personsskilled in the art upon review of the present application as a whole.The subject matter described herein and in the recited claims intends tocover and embrace all suitable changes in technology.

1-33. (canceled)
 34. A method for producing a physiological model of atleast one of a bone marrow and blood vessel structure, the methodcomprising: providing a biomimetic microfluidic system comprising: asubstrate; a first channel formed in the substrate, the first channelextending from a first input to a first output along a longitudinaldimension and a first transverse dimension; a second channel formed inthe substrate, the second channel extending from a second input to asecond output along the longitudinal dimension and the first transversedimension; a third channel formed in the substrate, the third channelextending from the second input to a third output along the longitudinaldimension and the first transverse dimension, wherein the first, second,and third channels extend substantially parallel along the longitudinaldimension and extend along a second transverse dimension; a series ofmicrochannels connecting the first channel to the second channel andconnecting the third channel to the first channel, wherein the series ofmicrochannels extend further in the longitudinal dimension than thefirst transverse direction and the second transverse direction and ispositioned proximal to a first portion of the substrate to create afluid communication path passing between the first channel and thesecond channel and the first channel and the third channel proximate tothe first portion of the substrate; a first source connected to thefirst input; a second source connected to the second input; introducinga first biological composition into the first channel at a first channelflow rate using the first source; introducing a second biologicalcomposition into the second channel and third channel using the secondsource and at a second channel flow rate and a third channel flow rate,respectively, to create a differential between the first, second andthird channel flow rates to generate physiological shear rates within apredetermined range in the channels; and harvesting a target biologicalsubstance produced proximate to the microchannels by the physiologicalshear rates.
 35. The method of claim 34, wherein each of the series ofmicrochannels is sized to capture of the plurality of blood plateletprogenitors generally within the series of microchannels.
 36. The methodof claim 34, wherein at least one of the first and second biologicalcompositions includes at least one of a semi-solid, a solid, a liquid,and a collection of cells, or a combination thereof.
 37. The method ofclaim 34, wherein the physiological shear rates facilitate a productionof a plurality blood platelets.
 38. The method of claim 34, wherein eachof the series of microchannels has dimensions between 0.1 micrometersand 20 micrometers.
 39. The method of claim 34, wherein the targetbiological substance comprises blood platelets.
 40. The method of claim34, wherein the biomimetic microfluidic system further comprises asecond channel port arranged upstream from the second input along thesecond channel, and a third channel port arranged upstream from thesecond input along the third channel.
 41. The method of claim 40 furthercomprising reversing a fluid flow direction in at least one of thefirst, second, and third channels using a combination of the firstinput, the first output, the second input, the second output, the thirdoutput, the second channel port, and third channel port.
 42. A methodfor producing a physiological model of at least one of a bone marrow andblood vessel structure, the method comprising: introducing, through afirst flow filter, a first biological composition into a first channelof a microfluidic system at a first channel flow rate, the firstbiological composition including a biological source material capable ofproducing a target biological substance; introducing, through a secondflow filter, a second biological composition into a second channel ofthe microfluidic system at a second channel flow rate; selectivelycapturing, by a membrane separating the first channel and the secondchannel and forming a fluid communication path between the first channeland the second channel, the biological source material from the firstbiological composition passing through the membrane; generatingphysiological shear rates on the captured biological source materialthat induce the captured biological source material to produce thetarget biological substance; and harvesting, using the second biologicalcomposition, the produced target biological substance from the secondchannel.
 43. The method of claim 42, wherein the microfluidic systemfurther comprises a substrate in which the first channel and the secondchannel are formed.
 44. The method of claim 42, wherein the firstchannel extends from a first input to a first output substantially alonga longitudinal direction and the second channel extends from a secondinput to a second output along the longitudinal direction, wherein atleast a portion of the first and second channels extends substantiallyparallel along the longitudinal direction.
 45. The method of claim 44,wherein the membrane separates the first channel and the second channelalong a transverse direction.
 46. The method of claim 44, wherein afirst flow resistor positioned between the first input and the firstflow filter; and a second flow resistor positioned between the secondinput and the second flow filter.
 47. The method of claim 42, whereingenerating physiological shear rates comprises adjusting the first flowrate and the second flow rate to create a differential between the firstchannel and the second channel that generates physiological shear ratesalong the second channel.
 48. The method of claim 42, wherein themembrane includes a plurality of pores sized less than the biologicalsource material to allow capture of the biological source materialgenerally about the pores.
 49. The method of claim 48, wherein the poreshave a diameter in a range between 3 micrometers and 12 micrometers. 50.The method of claim 42, wherein the physiological shear rates are in arange between 100 s−1 and 10,000 s−1.
 51. A method for producing aphysiological model of at least one of a bone marrow and blood vesselstructure, the method comprising: introducing, through a first flowfilter, a first biological composition into a first channel of amicrofluidic system at a first channel flow rate, the first biologicalcomposition including megakaryocytes (MKs) capable of generatingplatelets (PLTs); introducing, through a second flow filter, a secondbiological composition into a second channel of the microfluidic systemat a second channel flow rate; selectively capturing, by a membraneseparating the first channel and the second channel and forming a fluidcommunication path between the first channel and the second channel, theMKs from the first biological composition passing through the membrane;generating physiological shear rates on the MKs that induce the capturedMKs to produce the PLTs; and harvesting, using the second biologicalcomposition, the produced PLTs from the second channel.
 52. The methodof claim 51, wherein generating physiological shear rates comprisesadjusting the first flow rate and the second flow rate to create adifferential between the first channel and the second channel thatgenerates physiological shear rates along the second channel.
 53. Themethod of claim 51, wherein the physiological shear rates are in a rangebetween 100 s−1 and 10,000 s−1.